A major challenge facing biologists today is determining the function of over half a million partial cDNA sequences of various genes, known as expressed sequence tags (ESTs), that are publicly available. In most cases the function of the full-length genes represented by the ESTs remains unknown. Thus, the ability to determine function of these gene sequences is important for disease diagnosis, prediction, prevention and treatment.
In recent years, mouse geneticists have succeeded in creating transgenic animals by manipulating the genes of developing embryos and introducing foreign genes into these embryos. Once these genes have integrated into the genome of the recipient embryo, the resulting embryos or adult animals can be analyzed to determine the function of the gene.
U.S. Pat. Nos. 5,464,764 and 5,487,992 describe one type of transgenic animal in which the gene of interest is deleted or mutated sufficiently to disrupt its function. These “knock-out” animals are made by taking advantage of the phenomena of homologous recombination. (See, also U.S. Pat. Nos. 5,631,153 and 5,627,059). Briefly, conventional targeting DNA vectors contain (1) two blocks of DNA sequences that are homologous to separate regions of the target site; (2) a DNA sequence that codes for resistance to the compound G418 (Neor) between the two blocks of homologous DNA (i.e. positive selection marker) and (3) DNA sequences coding for herpes simplex virus thymidine kinases (HSV-tk1 and HSV-tk2) outside of the homologous blocks (i.e. negative selection marker). When this vector is introduced into the embryonic stem cell, homologous recombination inserts the Neor gene into the target genome, disrupting function of that gene.
The production of constructs useful in producing knock-out animals is a time and labor intensive process. (See, e.g., U.S. Pat. No. 5,464,764) First, genomic clones must be isolated by screening a genomic library with a radioactive probe. To isolate an individual clone requires multiple screens and can take more than 3 weeks. Once the clone is isolated, a restriction map is created in order to aid in the identification of fragments flanking the gene of interest. Again, this process can take several weeks. Finally, the flanking sequences are cloned into the targeting vector. Even in methods which make use of polymerase chain reaction techniques, a partial restriction map of the gene locus is created. (See, Randolph et al. (1996) Transgenic Research 5:413-420). In all, using conventional techniques, production of a DNA targeting construct can take several months.